Blood coagulation inhibiting proteins, processes for preparing them and their uses

ABSTRACT

This invention discloses proteins which inhibit the coagulation of the blood, processes for preparing these proteins, and the use thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to proteins which inhibit the coagulation of theblood, processes for preparing these proteins, and their use.

2. Description of the Background Art

Anti-coagulant proteins, which are present in most mammals, can bedivided into three groups based on their different mechanisms ofactivity.

One group of proteins form a complex with a coagulation factor andthereby render the coagulation factor inactive. Proteins in thiscategory include antithrombin III (Thromb. Res., 5: 439-452 (1974)),alpha₁ -protease inhibitor (Ann. Rev. Biochem., 52: 655-709 (1983)),alpha₂ -macroglobulin (Ann. Rev. Biochem., 52: 655-709 (1983)), C₁-inhibitor (Biochemistry, 20: 2738-2743 (1981)), and protease nexin (J.Biol. Chem., 258: 10,439-10,444 (1983)).

A second group of proteins act proteolytically on a coagulating factorand thereby inactivate it. The only protein of this kind that has beendescribed is protein C (J. Biol. Chem., 251: 355-363 (1976)).

The third category to which anti-coagulant proteins can be grouped arethose which screen and/or hydrolyze the negatively charged phospholipidsso that the phospholipid-dependent reactions of the blood coagulationmechanism are inhibited. Thus far, only phospholipases isolated fromvarious types of snake venom have been described as having this mode ofaction (Eur. J. Biochem., 112: 25-32 (1980)).

In recent years, the step-wise coagulation system has been investigatedthoroughly. It is understood to be an intensifying multi-stage system ofdifferent interconnected proteolytic reactions in which an enzymeconverts a zymogen into the active form (cf. Jackson, C. M. andNemerson, Y., Ann. Rev. Biochem., 49: 765-811 (1980)). The speed of thisreaction is decisively increased by the presence of phospholipids andother cofactors such as factor V_(a) and factor VIII_(a). In vivo, theprocoagulation reactions are regulated by a variety of inhibitorymechanisms which prevent an explosively thrombotic trauma after slightactivation of the coagulation cascade.

The mechanisms by which the anti-coagulation proteins of these threegroups act have been described (Rosenberg, R. D. and Rosenberg, J. S.,J. Clin. Invest., 74: 1-6 (1984)).

In Group 1, serine-protease factor X_(a) and thrombin are inactivated asa result of their binding to antithrombin III or to theantithrombin/heparin complex. Both the prothrombin activation and alsothe formation of fibrin can be inhibited in this way. In addition toantithrombin III, there are also various other plasmaprotease inhibitorssuch as alpha₂ -macroglobulin and antitrypsin, the activity of which isdependent on time.

In Group 2, the discovery of protein C led to another anti-coagulationmechanism. Once protein C is activated, it acts as an anti-coagulant byselective proteolysis of the protein cofactors V_(a) and VIII_(a), bywhich prothrombinase and the enzyme which converts factor X aredeactivated.

In Group 3, plasmin cleaves monomeric fibrin 1, a product of the effectof thrombin on fibrinogen, thereby preventing the formation of aninsoluble fibrin (Nossel, H. L., Nature, 291: 165-167 (1981)).

Of the above-mentioned native proteins involved in the coagulationprocess, at present only antithrombin III is clinically used. However,the increase in the tendency to bleed when this protein is administeredhas proven to be a serious disadvantage.

All the agents previously used as anticoagulants, whether native to thebody or synthetic, in some way render the coagulation factorsineffective and thereby lead to side effects which have adisadvantageous effect on the coagulation process.

SUMMARY OF THE INVENTION

It has been found possible to isolate native proteins which have bloodcoagulation-inhibiting properties, but do not increase the risk ofbleeding. These proteins lose their inhibiting properties in the eventof major bleeding, so that the normal coagulation processes can proceedwithout disruption and there is no danger of bleeding to death.

The present invention thus relates to anti-coagulant proteins,hereinafter referred to as VAC (Vascular Anti-Coagulant), which do notinactivate the coagulation factors. These proteins are capable ofinhibiting the coagulation induced by a vascular procoagulant or by thefactor X_(a), but do not inhibit the coagulation induced by thrombin. Inaddition, they do not inhibit the biological and amidolytic activity offactors X_(a) and II_(a).

DESCRIPTION OF THE FIGURES

FIG. 1: Gel Filtration of VAC on Sephadex G-100

The column (3×80 cm) was prepared at 60 cm pressure and equilibratedwith 500 mM NaCl and 20 mM Tris/HCl, pH 7.5. The VAC-containing fractionobtained after DEAE chromatography was concentrated (2 ml) and thenpassed over the Sephadex G-100. The pressure was maintained at 60 cm.and the void volume was 245 ml (fraction 70). The fractions (2 ml) weredialyzed against Tris-buffered saline (TBS) containing 10% glycerol, andtested for VAC activity by the one-stage coagulation test as describedin Example 1. The coagulation times were determined using 1:10 dilutionsof the G-100 fractions in TBS. The coagulation time in the absence ofVAC was 65 seconds.

FIG. 2: Analytical SDS-PAGE of VAC

SDS-PAGE gels contained by weight 10% acrylamide, 0.27% of N,N³-methylene-bisacrylamide, and 0.1% SDS (Laemli, U.K., Nature, 227:680-685 (1970)).

Lane 1: reduced reference proteins;

Lane 2: 25 ug reduced VAC;

Lane 3: 25 ug non-reduced VAC.

The gel was stained with Coomassie Blue and decolorized in the mannerdescribed in Example 1.

FIG. 3: Isoelectric pH of VAC

Electrofocusing was carried out with PAG plates in a pH range of from3.5-9.5 (see Example 1). 200 ug of human H_(b) ¹ and 20 ug of VAC wereapplied to the gel after the pH gradient had formed in the gel. HumanH_(b) was used as a reference (isoelectric point: pH 6.8). The gel wasfixed for 30 minutes with 0.7M trichloroacetic acid and stained withCoomassie Blue.

FIG. 4: Analysis of the Binding of VAC to Negatively ChargedPhospholipid Liposomes with SDS-PAGE

SDS-PAGE was carried out according to Laemli (Laemli, U.K. Nature, 227:680-685 (1970)) on the same plates as described in Example 1. Thesamples analyzed were obtained from the binding experiments as mentionedin the explanation to Table B.

Lane 1: reduced reference proteins; Lane 2: supernatant of VACpreparation centrifuged in the absence of liposomes; Lane 3: supernatantof VAC preparation centrifuged in the presence of liposomes; Lane 4:supernatant of VAC preparation centrifuged in the presence of liposomesand Ca⁺⁺ ; Lane 5: supernatant from liposome precipitate of Lane 4resuspended in TBS (10 mM EDTA) and centrifuged.

FIG. 5: Effect of VAC Concentration on Inhibition (%) of ThrombinFormation

The concentrations of VAC mentioned are the final concentrations presentin the test systems. The thrombin formation was measured in 1 uMprothrombin, 10 nM factor X_(a) and 0.5M ( ) or 5.0M (••) phospholipidmembrane (PC/PS; 4:1, mol/mol) in 10 mM TBSA with CaCl₂. The reactionmixture was stirred with the specific quantities of VAC (Specificactivity: 1300 units/mg) for 3 minutes at 37° C. without prothrombin. Byadding prothrombin to the mixture, as in Example 1, the thrombinformation was initiated and the speed measured. The speed of thrombinformation in the absence of VAC was 3.3 nM II_(a) /min. ( ) or 10.9 nMII_(a) /min. (• •).

FIG. 6: Effect of Phospholipid Concentration on Inhibition (%) ofThrombin Formation by VAC

Thrombin formation was measured at 1 um prothrombin, 10 nM factor X_(a),10.7 ug/ml VAC (Specific activity: 1300 units/mg) and at variousconcentrations of phospholipid membrane (PC/PS; 4:1, mol/mol) in TBSA.Factor X_(a), VAC and phospholipid were stirred in TBSA for 3 minutes at37° C. The thrombin formation was initiated by adding prothrombin to thereaction mixture. The rate of thrombin formation was measured asdescribed in Example 1. The percent inhibition of thrombin formation (••) was measured for each phospholipid concentration with thecorresponding rate of thrombin formation in the absence of VAC ( ).

FIG. 7: Gel Filtration of the 10,000×g Supernatant of an Umbilical CordArtery Homogenate on Sephadex G-100

2 ml of the 10,000×g supernatant of a homogenized umbilical cord wasloaded on a Sephadex G-100 column (1.5×80 cm), which waspre-equilibrated with TBS. The column was eluted with TBS. Aliquots ofthe resulting fractions were tested in the MPTT. Certain fractions ( )express a procoagulant activity and initiated coagulation in the MPTTwithout the addition of HTP, factor X_(a), or thrombin. Other distinctfractions ( ) prolong clotting time in the MPTT, using HTP to initiatecoagulation. These fractions were pooled and further fractionated.

FIG. 8: Chromtography of the Anti-Coagulant on DEAE-Sephacel (A) andSephadex G-75 (B).

The pool, containing the anti-coagulant, from the Sephadex G-100 columnwas applied to DEAE-Sephacel. Elution was performed with a 200 ml lineargradient of 50-300 mM NaCl (- - -). Fractions (4 ml) were collected.A₂₈₀ was measured for each fraction (--) and anti-coagulant activityassayed in the MPTT using HTP (final concentration: 95 ug protein/ml) asinitiator of coagulation (•). The fractions with anti-coagulant activitywere pooled, concentrated, and subsequently applied to Sephadex G-75(B). Fractions (2 ml) were collected. Each fraction was measured forA₂₈₀ (--) and anti-coagulant activity (•). V_(o) represents the voidvolume of the column.

FIG. 9: Dose Response of the Anti-Coagulant in the MPTT

Varying amounts of the anti-coagulant were added to the MPTT.Coagulation was initiated with HTP (final concentration: 95 ugprotein/ml). Control clotting time was 65 s.

FIG. 10: Gel Electrophoresis of Several Fractions of the G-75 Eluant

Several fractions of the G-75 eluant were analyzed by SDS-PAGE. The gelswere silver-stained according to Merril et al., Electrophoresis J., 3:17-23 (1982)). Lane 1: reduced low molecular weight standards; Lanes2-6: unreduced aliquots of the G-75 fractions numbers 35, 39, 41, 43 and50, respectively.

FIG. 11: The Effect of Proteolytic Enzymes on the Activity of theAnti-Coagulant

The anti-coagulant was incubated at 37° C. with protease type I ( ,final concentration: 0.11 units/ml), trypsin ( , final concentration: 88BAEE units/ml) and without proteolytic enzymes (•). At the timesindicated, 5 ul containing 6 ug protein of the anti-coagulant wasremoved from the reaction mixture and added to the MPTT. Clotting wasinitiated with HTP (final concentration: 18 ug protein/ml). Controlclotting time was 110 s. The units given in this legend for theproteolytic enzymes are calculated from the values supplied by themanufacturer.

FIG. 12: Effect of Vascular Anti-Coagulant on the Clotting Times,Induced in the MPTT by Either HTP, Factor X_(a), or thrombin

The concentrations of the coagulation initiators (HTP: 18 ug protein/ml,1.5 nM factor X_(a), or 0.4 nM thrombin) were chosen to give controlclotting times of about 110 seconds (open bars). When factor X_(a) wasused, phospholipid vesicles (final concentration 10 uM), composed of theOle₂ Gro-P-Ser/Ole₂ Gro-P-Cho (molar ratio, 20:80) were added to thereaction mixture. Clotting times induced by the indicated agents in thepresence of 3.5 ug anti-coagulant protein are shown by the shaded bars.

FIG. 13: Effect of the Anti-Coagulant of Prothrombin Activation by(X_(a), V_(a), phospholipid, CA²⁺), (X_(a), phospholipid, Ca²⁺), (X_(a),Ca²⁺).

The reaction mixtures contained: (A) 1 uM prothrombin, 0.3 nM X_(a), 0.6nM V_(a), 0.5 uM phospholipid and 10 mM CaCl₂ with 12.0 ug/mlanti-coagulant ( ), 4.8 ug/ml anticoagulant ( ), and 0.0 anticoagulant(•); (B) 1 uM prothrombin, 10 nM X_(a), 0.5 uM phospholipid, and 10 mMCaCl₂ with 2.4 ug/ml anti-coagulant ( ), 0.48 ug/ml anti-coagulant ( ),and 0.0 anti-coagulant (•); (C) 1 uM prothrombin, 75 nM X_(a), and 10 mMCaCl₂ with 120 ug/ml anti-coagulant ( ), and 0.0 anti-coagulant (•). Attimes indicated, samples were removed and thrombin was determined.

FIG. 14: Immunoblots

Immunoblots were obtained by the procedure described in Example 5. Lane1: bovine aorta protein fraction with VAC-activity; Lane 2: bovine aortaprotein fraction with VAC-activity; Lane 3: bovine lung protein fractionwith VAC-activity; Lane 4: human umbilical cord artery protein fractionwith VAC-activity; Lane 5: rat aorta protein fraction with VAC-activity;and Lane 6: horse aorta protein fraction with VAC-activity.

FIG. 15: Gel Electrophoresis (A) and Anti-Coagulant Activity (B) of theVarious Fractions of the G-75 Eluate

Various fractions of the G-75 eluate were subjected to gelelectrophoresis as described. The bands were stained with silver by themethod of Merril et al., Electrophoresis J., 3: 17-23 (1982).Electrophoresis lane 1: low molecular weight standards; electrophoresislanes 2-6: equal volumes of non-reduced G-75 fractions with increasingelution volume. Specific quantities of the G-75 fractions, which hadbeen analyzed by gel electrophoresis, were tested in the MPTT using HTPto initiate coagulation. The control coagulation time is represented bythe open bar. The figures under the shaded bars correspond to thenumbers of the electrophoresis lanes in FIG. 15A.

FIG. 16: Heat Inactivation of the Vascular Anti-Coagulation Agent (VAC)

The anti-coagulation agent was incubated at 56° C. and, after thevarious incubation periods, 5 ul samples containing 3.6 ug protein weretaken, immediately cooled with ice, and tested in the MPTT using HPT ascoagulation initiator. The coagulation time of the control sample was110 seconds.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to an agent which has bloodcoagulation-inhibiting properties but not the disadvantageous sideeffects on the coagulation process which accompany the anti-coagulantscurrently known.

The anti-coagulant proteins of the invention do not deactivate thecoagulation factors, but inhibit:

the modified prothrombin-time experiment and/or

the modified activated partial thromoplastin-time experiment and/or

the non-modified prothrombin-time experiment and/or

the prothrombin activation by the coagulation factor X_(a) in thepresence of negatively charged phospholipids and Ca²⁺ and/or

the intrinsic X-activation by factor IX_(a) in the presence ofnegatively charged phospholipids and Ca²⁺ and/or

the prothrombin activation of isolated stimulated blood platelets and/or

the coagulation induced by the walls of the blood vessels and/or

the coagulation-dependent platelet aggregation.

The invention also relates to anti-coagulant proteins that do notinactivate the coagulation factors and whose inhibitory activity dependson the concentration of phospholipids. The proteins of the inventioninduce inhibition of prothrombin activation by factor X_(a). Thisinhibition depends on the phospholipid concentration and is less at highphospholipid concentrations. Phospholipids are not hydrolyzed by theproteins of the invention.

The invention further relates to anti-coagulant proteins which do notinactivate the coagulation factors and which bind, via the divalentcations Ca²⁺ and/or Mn²⁺, to negatively charged phospholipids, which canbe found, for example, in vesicles, liposomes or etherosomes and/or, viathe divalent cations Ca²⁺ and/or Mn²⁺, to negatively chargedphospholipids which are coupled with Spherocil. The binding of theanticoagulant proteins of the invention to negatively chargedphospholipids is reversible and can be reversed by ethylenediaminetetraacetic acid (EDTA). The proteins according to the invention arecapable of displacing factor X_(a) and prothrombin from a negativelycharged phospholipid surface.

The invention relates particularly to anti-coagulant proteins which donot inactivate the coagulation factors and have molecular weights ofapproximately 70×10³, 60×10³, 34×10³, or 32×10³, of which the proteinswith a molecular weight of 34×10³ or 32×10³ have a single polypeptidechain.

The invention preferably relates to a family of anti-coagulant proteinswhich do not inactivate the coagulation factors and are characterized inthat:

they are isolated from the walls of blood vessels in mammals and arethen substantially purified,

they are not glycoproteins,

they are not phospholipases,

they have an isoelectric point of pH 4.4-4.6,

the activity of the anti-coagulant proteins at 56° C. is thermallyunstable,

the activity of the anti-coagulating proteins in citrated plasma remainsstable for some hours at 37° C.,

the activity of the anti-coagulant proteins is not completely destroyedby trypsin and/or chymotrypsin,

the activity of the anti-coagulant proteins is not affected bycollagenase and/or elastase,

they bind, via the divalent cations Ca²⁺ and Mn²⁺, to negatively chargedphospholipids which can be found in vesicles, liposomes or etherosomes,

they bind via the divalent cations Ca²⁺ and Mn²⁺ to negatively chargedphospholipids which are coupled to Spherocil,

the binding of the proteins to the negatively charged phospholipids isreversible and can be removed by ethylenediamine tetracetic acid (EDTA),

they displace factor X_(a) and prothrombin from a negatively chargedphospholipid surface,

they inhibit the modified thrombin-time experiment,

they inhibit the modified, activated, partial thromboplastin-timeexperiment,

they inhibit the non-modified prothrombin-time experiment,

they inhibit prothrombin activation by the coagulation factor X_(a) inthe presence of negatively charged phospholipids and Ca²⁺ in vitro,

they do not inhibit the biological and amidolytic activity of factorsX_(a) and II_(a),

they inhibit the intrinsic X-activation by the factor IX_(a) in thepresence of negatively charged phospholipids and Ca²⁺ in vitro,

they inhibit the prothrombin activation of isolated, stimulated bloodplatelets in vitro,

they inhibit the coagulation induced by the walls of the blood vesselsin vitro, and

the inhibition of prothrombin activation by factor X_(a) induced by theproteins is dependent on the concentration of phospholipids and isreduced at high phospholipid concentrations.

In particular, the invention relates to VAC proteins substantially freeof any animal tissue, especially in substantially pure form.

Suitable starting materials for the isolation of the VAC proteins arethe blood vessel walls and highly vascularized tissue of variousmammals, for example, cattle, rats, horses, and humans, as well asendothelial cell cultures of these mammals. The arterial walls ofcattle, rats, horses, and humans and human umbilical veins and arteriesare particularly suitable.

The invention also relates to a process for preparing the proteins ofthe invention using isolation and purification techniques. In aprocedure which is particularly suitable, the starting material ishomogenized and subjected to differential centrifugation. Thesupernatant liquid obtained can then be further treated as follows inany desired sequence. Undesirable contaminants can be precipitated withammonium sulfate. The supernatant is then further purified by affinitychromatography, for example, using hydroxyapatite; ion exchangechromatography, for example, using DEAE-Sephacel; chromatograpy over amolecular sieve, such as Sephadex G-100, and immunoabsorptionchromatography, for example, with polyclonal or monoclonal antibodies.Depending on the quality of the starting material the purificationprocess can be modified or other purification procedures can be usedsuch as, for example, phospholipid vesicles.

In addition to the classic anti-thrombosis treatment, namely, coagulantstaken orally, more recently biosynthetic tissue-plasminogen activatorhas been administered by the intrasvascular route for cases of manifestthrombosis (N. Engl. J. Med., 310: 609-513 (1984)).

The proteins according to the present invention are especially suitablefor preventing thrombosis, for example, during operations, because oftheir blood coagulation-inhibiting properties while at the same timeinhibiting the coagulation-dependent aggregation of platelets.

The present invention therefore also relates to the use of the proteinsaccording to the invention as antithrombotic agents.

The invention further relates to pharmaceutical compositions whichcomprise at least one protein according to the invention in associationwith a pharmaceutically acceptable carrier and/or excipient.

The anti-coagulant proteins of the invention can be administeredparenterally by injection or by gradual perfusion over time. They can beadministered intravenously, intraperitoneally, intrasmuscularly, orsubcutaneously.

Preparations for parenteral administration include sterile or aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate.

Aqueous carriers include water, alcoholic/aqueous solutions, emulsionsor suspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers, such as thosebased on Ringer's dextrose, and the like. Preservatives and otheradditives can also be present, such as, for example, antimicrobials,antioxidants, chelating agents, inert gases, and the like. See,generally, Remington's Pharmaceutical Science, 16th Ed., Mack, eds.1980.

The invention also relates to a method for preparing a medicament orpharmaceutical composition comprising the components of the invention,the medicament being used for anti-coagulant therapy.

Results from the isolation and purification of VAC from bovine arteriesare shown in Table A. Determination of the level of VAC activity in thesupernatant of the 100,000×g centrifugation was erroneous owing to thepresence of procoagulant activity. The components responsible for thisactivity were found to be precipitated with ammonium sulfate at asaturation level of 35%. It was discovered that the supernatant solutionobtained after precipitation with 35% ammonium sulfate contained 100%VAC activity. In order to precipitate this activity, the solution wasmixed with ammonium sulfate until 90% saturation was achieved. Theresulting precipitate containing the VAC proteins was bound to ahydroxyapatite column in the presence of TBS (100 mM NaCl, 50 mMTris/HCl, pH 7.5). After washing, the VAC proteins were eluted from thiscolumn with an increasing phosphate gradient. At low ion concentration,the VAC proteins were bound to the DEAE-Sephacel column. Elution of theVAC proteins from this column was done using an increasing NaClconcentration gradient. In the final purification step, the proteinswere separated on the basis of their molecular weight by gel filtrationon Sephadex G-100. A high-salt buffer was used as the eluant to minimizethe interaction of VAC with the Sephadex material. VAC was eluted fromthis column in a volume of about 1.6 times the void volume of the column(see FIG. 1). The total yield of VAC after this final purification was35%. By SDS-PAGE, all G-100 fractions which showed VAC activity werefound to contain two polypeptides (molecular weight 34,000 and 32,000,respectively). In some cases, an additional fraction with a molecularweight of 60,000 showed VAC-activity.

Using SDS-PAGE, only peak fractions 138-140 were homogeneous in relationto the two polypeptides. These fractions were used for all otherexperiments concerning investigation of bovine VAC described in thespecification, with the exception of the experiments for characterizingthe binding of bovine VAC to phospholipid liposomes.

In G-100 fraction 139, 3.4% of the VAC activity was found to have aspecific activity of 1480 units per mg of protein by means of aone-stage coagulation test (see Example 1 and Table A). This fractioncontained no detectable quantity of phospholipid. An extinctioncoefficient of ##EQU1## was calculated for this purified VAC preparationfrom the absorption at 280 nm and from the protein content.

As shown in FIG. 2, the two polypeptides with molecular weights of34,000 and 32,000, which are present in the purified protein materialfrom bovine arteries and to which VAC activity has been ascribed, have asingle polypeptide chain. Using Schiff's reagent with basic fuchsin, itwas established that both proteins contain few carbohydrate groups.Moreover, no gamma-carboxyglutamate (Gla) residues could be found ineither protein. Isoelectric focusing (Example 1) showed that bothproteins migrate in a single band corresponding to an isoelectric pointof 4.4 to 4.6 (FIG. 3).

The VAC activity was obtained from the PAG plate by elution of this bandfrom the gel. Analysis of the eluant with SDS-PAGE again showed thepresence of the two proteins. It was thus possible to confirm that bothproteins migrate in a single band in the pH gradient of the PAG plate.In order to check the method, human hemoglobin (Hb) was alsoinvestigated by isoelectric focusing. The value of 6.8 found for Hbagrees with the value given in the literature (see FIG. 3).

Binding experiments showed that the VAC activity can bind to negativelycharged phospholipid membranes. This binding takes place in the presenceof Ca²⁺ and Mn²⁺, but not in the presence of Mg²⁺ or in the absence ofdivalent metal ions (see Table B). This binding of VAC activity toliposomes is reversible using EDTA.

Using SDS-PAGE, it was possible to show that both proteins can bind toliposomes in the presence of Ca²⁺ and that this binding is disruptedwhen EDTA is added (see FIG. 4). This is yet another indication that VACactivity can be ascribed to these two proteins.

On storage in tris-buffered saline (TBS) containing 10% glycerol, VACactivity is stable at -70° C. for at least three months, at 0° C. for atleast 12 hours, and at 37° C. for at least half an hour. At 56° C., theactivity disappears within two minutes.

The activity of VAC prolongs the coagulation time in a one-stagecoagulation experiment (Example 1) in which coagulation is triggeredwith thromboplastin from bovine brains (BTP). Replacement of BTP in thisexperiment with purified bovine thrombin or purified bovine factor X_(a)showed that VAC prolongs the coagulation time only if factor X_(a) isused to initiate coagulation; coagulation induced by thrombin is notaffected by VAC. This indicates that VAC directly inhibits the factorX_(a) activity or that there is some interaction with the prothrombinasecomplex.

In further testing, an amidolytic thrombin formation test using purifiedbovine factor X_(a) and prothrombin was carried out. FIG. 5 shows thatwhen prothrombin is activated in the presence of Ca²⁺ and phospholipidby means of factor X_(a) to form thrombin, VAC inhibits the prothrombinactivation and the degree of inhibition is dependent on theconcentration of VAC. Moreover, the inhibiting effect of VAC is greaterat a lower concentration of phospholipid.

FIG. 6 shows the phospholipid dependency of the VAC-induced inhibitionof prothrombin activation. It is significant that at a phospholipidconcentration of zero the prothrombin activation by factor X_(a) is notinhibited by VAC. Control tests showed that VAC itself has no effect onthe system of measurement.

Incubation of 5 uM phospholipid [1,2-dioleoylsn-glycero-3-phosphoserine(PS)/1,2-dioleoyl-sn-glycerol-3-phosphocholine (PC), 1:4 mol/mol] with107 ug/ml VAC (specific activity: 1,300 units per mg) and 10 mM Ca²⁺reduced the procoagulant activity within 3 minutes at 37° C. This showsthat VAC has no phospholipase activity.

In contrast to antithrombin III (AT-III), VAC has no effect on theamidolytic activity of purified thrombin and no lasting effect on factorX_(a) activity, as measured with the chromogenic substrate S 2337(N-benzoyl-L-isoleucyl-L-glutamyl-L-pipecolyl-glycyl-L-arginine-p-nitroanilide-dihydrochloride)or S 2238(H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide-dihydrochloride)[see Table C]. This table also shows that the inactivation of factorX_(a) and thrombin by AT-III is not intensified by VAC. Heparin, on theother hand, decisively increases inactivation of thrombin and factorX_(a) in the presence of AT-III. This shows that VAC has neither aheparin-like activity nor at AT-III-like activity.

The isolation of the anti-coagulant of the invention from human tissuemay be achieved by the same isolation procedure using, for example, ahomogenate of human umbilical cord arteries. In such an homogenate, ananti-coagulant according to the present invention has been discovered byits ability to prolong the clotting time in a prothrombin time test. Theanti-coagulation activity became measurable after Sephadex G-100fractionation of the arterial homogenate [See Example 4]. From furtherisolation procedures, this activity is associated with a water-solublesubstance(s), that carries an overall negative charge at pH 7.9.

Analysis of Sephadex G-75 fractions with gel electrophoresis has shown apositive correlation between the intensity of the 32,000 MW band and theprolongation of the clotting time as measured with a modifiedprothrombin time test (MPTT) [See Example 4]. The connection between the32K-band and anti-coagulant activity is demonstrated by the fact thatonly the 32K-band of the polyacrylamide gel has anti-coagulant activity.In combination with the findings that the anti-coagulant rapidly losesits activity when incubated at 56° C., and that proteolytic enzymes candestroy its activity, it is likely that the anti-coagulant activity isexpressed by a single protein with a molecular weight of 32,000 daltons.

Trypsin, in contrast to protease type I, is a poor inactivator of theanti-coagulant. This suggests that the anti-coagulant possesses only asmall number of lysine- and arginine-residues that are accessible totrypsin. The nature of the anti-coagulant activity has been studied byinitiating coagulation in different ways. Clotting, induced by eitherthe vascular procoagulant, HTP (human brain thromboplastin), or factorX_(a), is inhibited by the anti-coagulant; thrombin-induced clotting, onthe other hand, is not. From these findings, one can conclude that theanti-coagulant interferes with thrombin formation, not with thrombinaction.

Prothrombinase reconstituted from purified factors and prothrombin wereused to further study the anti-coagulant mechanism [See Example 4].Under these experimental conditions, the anti-coagulant can inhibit theactivation of prothrombin by complete prothrombinase (factor X_(a),factor V_(a), phospholipid, Ca²⁺) and by phospholipid-bound factor X_(a)(factor X_(a), phospholipid, Ca²⁺) but not by free factor X_(a) (factorX_(a), Ca²⁺).

The time course for prothrombin activation in the presence of theanti-coagulant indicate an instantaneous inhibition of prothrombinactivation which remains constant in time. This shows that theanti-coagulant acts neither by a phospholipase, nor by a proteolyticactivity. The fact that the activation of prothrombin by factor X_(a)and Ca²⁺ is not affected by the anti-coagulant at all, stronglyindicates that the anti-coagulant mechanism of the vascular compounddiffers from that of the well known plasma protease inhibitors such asantithrombin III. Since Walker et al., Biochim. Biophys. Acta, 571:333-342 (1979), have demonstrated that activated protein C does notinhibit prothrombin activation by factor X_(a), Ca²⁺ and phospholipid,it can also be concluded that this compound is not protein C and doesnot belong in Group 2 described above.

Preliminary binding studies indicate that the vascular anti-coagulantprobably interferes with the lipid binding of factor X_(a) and/orprothrombin. Whether the ability of the anti-coagulant to inhibitprothrombin activation completely accounts for its prolongation of theprothrombin time remains to be established.

The fact that this inhibitor can be found in various types of arteries,but not in poorly vascularized tissue indicates that a physiologicalmodulator of hemostasis and thrombosis, active at the vascular level,has been found.

On the absis of the properties and activities of VAC which have beenobserved, the blood coagulation mechanism under the influence of VAC maybe interpreted.

VAC binds via Ca²⁺ ions to negatively charged phospholipids which occuras a result of damage to the tissues and/or because of the stimulationof blood platelets, and thereby reduces the binding of specificcoagulation factors (vitamin K-dependent coagulation factors) to thenegatively charged phospholipid surface which acts as a catalyticsurface for these coagulation factors (Biochem. Biophys. Acta, 515:163-205 (1985)). As a result, the phospholipid-dependent bloodcoagulation reactions are inhibited by VAC. On the basis of itsmechanism of activity, VAC can be categorized in Group 3 describedabove.

However, a critical difference between VAC and the other known proteinsof this group is that VAC does not hydrolyze phospholipids and thereforedoes not decompose any essential membrane structures.

Among the properties of VAC which have not hitherto been described forany of the known anti-coagulants is the fact that the anti-coagulationeffect of VAC is dependent on the concentration of phospholipids in thecoagluation process. This dependency means that the coagulation processwhich has been initiated, for example, by slight damage to the wall ofthe blood vessel and/or by slight activation of blood platelets, thatis, by a thrombotic process, can be inhibited by VAC. On the other hand,the coagulation process which is triggered by severe damage to walls ofblood vessels, wherein phospholipids are present in high concentrations,is not inhibitied by VAC, because of high phospholipid concentrations.The danger of severe bleeding when using VAC is therefore extremelysmall. This property of VAC is in contrast to all the previously knownanti-coagulants which render one or more of the coagulating factorsineffective and thereby increase the risk of severe bleeding.

Another surprising property of VAC is that it does not deactivate thecoagulating factors themselves. Consequently, the coagulating factorscan still perform their other functions. For example, some activecoagulating factors also play a non-hemostatic role in the chemotaxis ofthe inflammatory cells which participate in the repair of damaged bloodvessel walls.

This invention further describes a novel class of anti-coagulantproteins which do not inactivate the coagulation factors. The Examplesserving to illustrate the invention and the properties listed should notrestrict the invention in any way. Anyone skilled in the art will beable, without any inventive effort, to obtain other proteins which haveanti-coagulant properties without inactivating the coagulation factors,using the method described. These proteins also fall within the scope ofprotection of this invention.

The abbreviations used in the invention have the following meanings:

VAC: vascular anti-coagulant

PFP: platelet free plasma

TBS: 100 mM NaCl, 50 mM Tris/HCl, pH 7.5

EDTA: ethylenediamine tetraacetic acid

TBSE: TBS with 2 mM of EDTA

BTP: thromboplastin from bovine brains

HTP: thromboplastin from human brains

TBSA: TBS with 0.5 mg/ml of human serum albumin, pH 7.9

S 2337:N-benzoyl-L-isoleucyl-L-glutamyl-L-pipecolyl-glycyl-L-arginine-p-nitroanilide-dihydrochloride

S 2238:H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide-dihydrochloride

AT-III: human antithrombin III

S.A.: specific activity

Ole₂ Gro-P-Cho: 1,2,-dioleolyl-sn-glycero-3-phosphocholine

Ole₂ Gro-P-Ser: 1,2,-dioleolyl-sn-glycero-3-phosphoserine

The nomenclature of the blood coagulation factors used was thatrecommended by the Task Force on Nomenclature of Blood Clotting Zymogensand Zymogen Intermediates.

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples which are includedherein for purposes of illustration only and are not intended to belimiting of the invention unless otherwise specified. Particularly, itis noted that, in principle, the present invention applies to allanti-coagulants from human and other animal sources, provided that theysatisfy the purity and reactivity criteria, and also to preparations ofthe above-described compounds obtained by methods other than thosedisclosed herein.

EXAMPLE 1 Characterization of VAC

(a) Isolation and Purification of VAC

The chemicals for analytical SDS-PAGE and hydroxyapatite (HTP) wereobtained from Bio-Rad. Sephadex G-100 and G-75, DEAE-Sephacel and the"Low Molecular Weight Calibration Kit" were obtained from Pharmacia. Thechromogenic substrates S 2337 and S 2238 were obtained from Kabi Vitrumand the Diaflo PM-10 ultrafiltration membrane was obtained from Amicon.

Bovine aortas were taken within half an hour after slaughtering theanimals. Bovine blood was collected in trisodium citrate (finalconcentration 0.38% by weight) and centrifuged for 10 minutes at ambienttemperature at 2,000×g. The plasma containing few blood platelets wasthen centrifuged again (15 minutes at 10,000×g). In this way,platelet-free plasma was obtained (PFP).

The aortas from the animals were thoroughly rinsed with TBS (100 mMNaCl, 50 mM Tris/HCl, pH 7.5) immediately after being removed. The innerlining of the aortas was removed and homogenized using a high-speedhomogenizer, e.g., the Braun MX 32, in TBSE (TBS with 2 mM EDTA)containing soyabean trypsin inhibitor (16 mg/l) and benzamidine (1.57g/l).

The material homogenized from eight aortas and containing 20% solids(weight/volume) was centrifuged for 60 minutes at 100,000×g. Thesupernatant was saturated with solid ammonium sulfate to 30% saturation,stirred from 30 minutes, and then centrifuged for 20 minutes at12,000×g. The resulting supernatant was saturated with solid ammoniumsulfate to 90% saturation, stirred for 30 minutes, and centrifuged for20 minutes at 12,000×g.

The precipitate was suspended in a small volume of TBS and dialyzed withTBS containing benzamidine (1.57 g/l). The dialyzed fraction was appliedto a hydroxyapatite column (1×20 cm) which had been equilibrated withTBS. The column was washed with four bed volumes of TBS and the VACproteins eluted with 200 ml of sodium phosphate buffer (pH 7.5) using alinear gradient (0-500 mM). The fractions containing VAC were combinedand dialyzed against 50 mM of NaCl with 20 mM of Tris/HCl at pH 7.5.

This same buffer was used to equilibrate a DEAE-Sephacel column (3×5 cm)on which the dialyzed VAC material was chromatographed. The column waswashed with four bed volumes of the equilibration buffer and the VACeluted with 200 ml of NaCl solution in 20 mM of Tris/HCl, pH 7.5, usinga linear gradient (50-300 mM). The fractions containing VAC werecollected, dialyzed with 500 mM NaCl in 20 mM of Tris/HCl at pH 7.5 andthen concentrated in an Amicon concentration cell using a PM-10ultrafiltration membrane. The concentrate (2 ml) was applied to aSephadex G-100 column (3×80 cm) equilibrated with 500 mM NaCl in 20 mMTris/HCl, pH 7.5.

The eluate was collected in 2 ml fractions and the active fractionsdialyzed separately against TBS containing 10% by volume glycerol andstored at -70° C. The entire purification was carried out at 0°-4° C.

b. Determining VAC Activity

Two different methods (see, generally, Harrison's Principles of InternalMedicine, 10th Ed., Petersdorf et al., eds., 1983) were used todetermine the VAC activity:

(a) the one-stage coagulation test (modified prothrombin time test)

(b) thrombin formation test.

The one-stage coagulation test was carried out as follows:

In a siliconized glass dish, 175 ul of the fraction to be tested, or 175ul of TBS as control, were stirred with 50 ul of PFP and 25 ul of diluteBTP (900 rpm). After incubation (3 minutes at 37° C.), coagulation wasinitiated by adding 250 ul of buffer which contained 80 mM NaCl, 20 mMCaCl₂, and 10 mM Tris/HCl, pH 7.5. Fibrin formation was recordedoptically using a "Payton Dual Aggregation Module" (Hornstra, G., Phil.Trans. R. Soc. London B, 294: 355-371 (1981)). The coagulation time ofthe control sample was 65 seconds. This test was used duringpurification to examine the various fractions for the presence of VACactivity. In order to determine the VAC yield during purification, oneunit of VAC activity was defined as the quantity of VAC which prolongsthe coagulation time in the above test to 100 seconds.

In some cases, BTP was replaced by purified bovine thrombin or thepurified bovine factor X_(a). In this semi-purified coagulation system,the quantity of thrombin or factor X_(a) used were such that thecoagulation time of the control sample was also 65 seconds.

The thrombin formation test was carried out as follows:

20 ul of purified bovine factor X_(a) (150 nM), 30 ul of CaCl₂ (100 mM),30 ul of dilute VAC and 30 ul of PS/PC-phospholipid membrane (the finalconcentrations are given in the legend accompanying FIG. 6) were placedin a plastic dish containing 181 ul TBSA (TBS with 0.5 mg/ml human serumalbumin, pH 7.9).

This mixture was stirred for 3 minutes at 37° C. with a Teflon stirrer.Thrombin formation was initiated by adding 9 ul of purified bovinefactor II (33.33 uM). At various times, 50 ul samples of the reactionmixture were added to a plastic dish containing 900 ul of TBSE and 50 ulof chromogenic substrate S 2238 (5 mM, 37° C.). The concentration ofthrombin in the reaction mixture was calculated from the change inextinction at 405 nm (Kontron Spectrometer Uvikon 810), using acalibration curve plotted from assays with known quantities of purifiedbovine thrombin. The percent inhibition caused by VAC was defined asfollows: ##EQU2## wherein "a" is the rate of thrombin formation in theabsence of VAC in nM II_(a) /min, and "b" is the rate of thrombinformation in the absence of VAC in nM II_(a) /min.

The vitamin K-dependent factors prothrombin and factor X_(a) wereobtained by purification of citrated bovine plasma (cf. Stenflo, J., J.Biol. Chem., 251: 355-363 (1976)). After barium citrate absorption andelution, fractionation with ammonium sulfate, and chromatography onDEAE-Sephadex, there were two protein fractions which contained amixture of prothrombin and factor IX or factor X. Factor X was activatedusing the method of Fujikawa et al., Biochemistry, 11: 4882-4891 (1972)and using RVV-X (Fujikawa et al., Biochemistry, 11: 4892-4899 (1972)).Prothrombin was separated from factor IX by heparinagarose affinitychromatography (Fujikawa et al., Biochemistry, 12: 4938-4945 (1973)).The prothrombin-containing fractions from the heparin-agarose columnwere combined and further purified using the method of Owens et al., J.Biol. Chem., 249: 594-605 (1974). The concentrations of prothrombin andfactor X_(a) were determined using the method of Rosing et al., J. Biol.Chem., 255: 274-283 (1980). BTP was prepared by the method of VanDam-Mieres et al., Blood Coagulation Enzymes, Methods of EnzymaticAnalysis, Verlag Chemie GmbH, Weinheim. The protein concentrations weredetermined according to Lowry et al., J. Biol. Chem., 193: 265 (1951).

C. Preparation of Phospholipids, Phospholipid Membranes and PhospholipidLiposomes

Phospholipids were prepared using1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1_(cis) /18:1_(cis) -PC)and 1,2-dioleoyl-sn-glycero-3-phosphoserine (18:1_(cis) /18:1_(cis)-PS), as described by Rosing et al., J. Biol. Chem., 255: 274-283(1980). Separate phospholipid membranes of PC and PS consisting of twolayers were prepared using ultrasound as described by Rosing et al., J.Biol. Chem., 255: 274-283 (1980). A supply of phospholipid liposomes wasprepared by dissolving the appropriate amount of phospholipid inchloroform which was evaporated using nitrogen. The residualphospholipid was suspended in TBS containg 5% glycerol, carefully mixedwith a few glass beads for 3 minutes, then centrifuged for 10 minutes at10,000 xg. The above solution was discarded and the residue carefullyresuspended in TBS containing 5% glycerol. In this manner, thephospholipid-liposome supply solution was obtained. These liposomes werestored at ambient temperature. The phospholipid concentration wasdetermined by phosphate analysis according to Bottcher et al., Anal.Chim. Acta., 24: 203-207 (1961).

Gel electrophoresis on plates in the presence of SDS was carried outaccording to the method described by Laemmli, Nature, 227: 680-685(1970) using a gel which contained 10% by weight acrylamide, 0.27% byweight N,N³ -methylene-bisacrylamide and 0.1% by weight SDS. In gelsamples with reduced disulfide bridges, 5% by weightbeta-mercapto-ethanol was present. The gels were stained as follows:

(1) 0.25% by weight Coomassie Blue R-250 in 50% by weight ethanol and15% by weight acetic acid, and decolorized with 10% by weight ethanoland 10% by weight acetic acid, or

(2) with Schiff's reagent prepared from basic fuchsin (Merck) by themethod of Segrest et al., described in Methods in Enzymology, Vol. 28,54-63 (1972), or

(3) with silver as described by Merril et al. in Electrophoresis, 3:17-23 (1982).

The isoelectric pH measurements of proteins were done using thin layerpolyacrylamide gels which contain ampholine carrier ampholyte (PAGplates, LKB) at a pH range of 3.5-9.5 in accordance with themanufacturer's instructions. The pH gradient in the gel was determinedimmediately after electrofocusing by cutting off a strip of the gelalong a line between the anode and the cathode. The electrolytes wereeluted from each strip using distilled water and the pH measured with acombined glass electrode.

The Gla determination was carried out by HPLC on a "Nucleosil 5SB"column (CHROMPACK) using the method of Kuwada et al., Anal. Biochem.,131: 173-179 (1983).

EXAMPLE 2 Coupling of Phospholipids to Spherocil

The required phospholipids were dissolved in chloroform and added to thecolumn material (Spherocil, Messrs. Rhone-Poulenc) at a ratio of 5 mg ofphospholipid per gram of Spherocil. The chloroform was evaporated withN₂ gas and the dry Spherocil phospholipid was then washed with thebuffer in which VAC had been suspended. VAC binds to Spherocil-coupledphospholipid in the presence of Ca⁺⁺ and/or Mn⁺⁺ when some of thephospholipids are negatively charged.

EXAMPLE 3

50 ul of citrated/platelet-free plasma were mixed with 200 ul of buffer(25 mM Tris/HCl, pH 7.5, 100 mM NaCl), containing kaolin (catalyzescoagulation), inositin (phospholipid source) and VAC were present. Thismixture was incubated (3 minutes at 37° C.) and 250 ul of Ca⁺⁺ buffer(200 mM Tris/HCl, pH 7.5, 80 mM NaCl, 20 mM CaCl₂) was added. Thecoagulation time was measured as described in Example 1.

EXAMPLE 4 Isolation and Characterization of Anti-Coagulant From HumanTissue

Human blood was collected by venipuncture in trisodium citrate (13 mM)and centrifuged at 2,000 xg for 10 minutes at room temperature. Theresulting plasma was recentrifuged at 1,000 xg for 15 minutes in orderto obtain platelet free plasma (PFP). A standard pool of PFP wasprepared by mixing plasma from several healthy donors.

Human umbilical cords were obtained within 15 minutes after delivery.The arteries were immediately perfused with ice-cold TBS-buffer,subsequently prepared free from the Jelly of Warton, and homogenized inTBS using a whirl mixer (Braun MX32). A 10% homogenate (w/v) was thenfractionated.

Fractionation of the supernatant from a 10,000 xg centrifugation of thehomogenate on Sephadex G-100 results in a reproducible profile (see FIG.7). The fractions affecting the coagulation system as measured with theMPTT are indicated in FIG. 7. Procoagulant activity eluted with the voidvolume. This activity can only be detected in the presence of factor VIIin the MPTT, as indicated by experiments in which human congenitalfactor VII-deficient plasma was used. This establishes that thisprocoagulant is tissue thromboplastin.

Certain fractions showed a distinct anti-coagulant activity. Thesefractions were pooled and further purified with DEAE-Sephacelchromatography (see FIG. 8A). The anti-coagulant bound to theDEAE-Sephacel with 50 mM NaCl in 50 mM Tris/HCl, pH 7.9. Elution ofactivity with a linear gradient of NaCl at pH 7.9 was achieved at150-160 mM NaCl. The DEAE-fractions expressing anti-coagulant activitywere pooled and filtered using Sephadex G-75 (FIG. 8B). The column(1.5×50 cm) was equilibrated with TBS and activity was present in thosefractions which corresponded to molecular weights of about 30,000-60,000daltons.

The MPTT was used as a quantitative assay for the determination of theamount of anti-coagulant activity (see FIG. 9). One unit ofanti-coagulant activity was defined as that quantity which prolongs theclotting time in the MPTT, with HTP (final concentration 95 ugprotein/ml) as initiator of coagulation, from its control value of 65 sto 100 s. With this assay, it was calculated that from 10 g wet arterialtissue 2 mg protein with approximately 1,200 units anti-coagulantactivity can be isolated.

The modified prothrombin time test (MPTT) was carried out as follows:

In a siliconized glass cuvette, 50 ul PFP was stirred at 37° C. with 150ul TBS, 25 ul of a standard HTP-dilution, and 25 ul TBS (control) or 25ul of a fraction of the arterial homogenate. After incubation for 3minutes, coagulation was started at time zero with the addition of 250ul Ca²⁺ -buffer (80 mM NaCl, 20 mM CaCl₂ and 10 mM Tris/HCl, pH 7.9).Fibrin formation was monitored optically (Payton Dual AggregationModule). When factor X_(a) was utilized to initiate coagulation in theMPTT, HTP was omitted and 25 ul purified factor X_(a) was added togetherwith the 250 ul Ca²⁺ -buffer to the diluted PFP.

The modified thrombin time test (MTT) was carried out similar to theX_(a) -initiated MPTT described above, with the exception that the X_(a)-preparation was replaced by 25 ul of purified thrombin.

Protease type I and trypsin (EC 3.4.2.1.4) were obtained from Sigma. HTPwas prepared from human brain as described by van Dam Mieras et al.,Methods of Enzymatic Analysis, 5: 352-365 (1984). Factor X_(a),prothrombin and thrombin were purified from citrated bovine blood asdescribed by Rosing et al., J. Biol. Chem., 255: 274-283 (1980). FactorV was purified from bovine blood as described by Lindhout et al.,Biochemistry, 21: 4594-5502 (1982). Factor V_(a) was obtained byincubating factor V with thrombin. Prothrombin concentrations werecalculated from MW=72,000 and A₂₈₀ ^(1%) =9.6 (Owen et al., J. Biol.Chem. 249: 594-605 (1974), and factor V concentration was calculatedfrom MW=330,000 and A₂₈₀ ^(1%) =9.6 (Nesheim et al., J. Biol. Chem.,254: 508-517 (1979). Factor X_(a) and thrombin concentrations weredetermined by active site titration (Rosing et al., J. Biol. Chem., 253:274-283 (1980). Other protein concentrations were determined asdescribed by Lowry et al., J. Biol. Chem., 193: 265 (1951).

Phospholipid and phospholipid vesicles were prepared using Ole₂Gro-P-Cho(1,2-dioleoyl-sn-glycero-3-phosphocholine) and Ole₂Gro-P-Ser(1,2-dioleoyl-sn-glycero-3-phosphoserine) as described inRosing et al., supra (1980). Single bilayer vesicles composed of Ole₂Gro-P-Ser/Ole₂ Gro-P-Cho (molar ratio 20:80) were prepared bysonication. Phospholipid concentrations were determined by phosphateanalysis according to the method of Bottcher et al., Anal. Chim. Acta,24: 203-207 (1961).

The time course of prothrombin activation was examined at differentconcentrations of anti-coagulant. Mixtures of (X_(a), Ca²⁺), (X_(a),phospholipid, Ca²⁺) or (X_(a), V_(a), phospholipid, Ca²⁺) were stirredwith different amounts of the anti-coagulant at 37° C. in 50 mMTris/HCl, 175 mM NaCl, 0.5 mg/ml human serum albumin at pH 7.9. After 3minutes, prothrombin activation was started by the addition ofprothrombin. At different time intervals, a 25 ul sample was transferredfrom the reaction mixture into a cuvette (37° C.), containing TBS, 2 mMEDTA and 0.23 mM S 2238 (final volume: 1 ml). From the absorption changeat 405 nm (Kontron Spectrophotometer Uvikon 810), and a calibrationcurve based on purified thrombin, the amount of thrombin formed wascalculated at different concentrations of anti-coagulant.

Phospholipid was added in the form of vesicles composed of Ole₂Gro-P-Ser and Ole₂ Gro-P-Cho with a molar ratio of 20:80.

Several fractions from G-75 chromatography were tested by MPTT andanalyzed using SDS-PAGE. The results (FIG. 10) showed that theanti-coagulant has a molecular weight of approximately 32,000 daltons.The anti-coagulant activity of the 32K-band was confirmed by slicing thepolyacrylamide gel, eluting the protein and testing the eluant foranti-coagulant activity as described above. Anti-coagulant activity wasfound only in the eluant in the slice corresponding to the 32K band.This activity was found to be stable at 56° C. and had a dose responserelationship in the MPTT similar to the starting material.

The G-75 fractions containing the highest anti-coagulant activity werepooled and used for further characterization of the anti-coagulant.Incubation of the anti-coagulant at 56° C. rapidly decreases itsactivity until after 2 minutes no activity can be measured. Theanti-coagulant loses its activity completely within 2 hours uponincubation at 37° C. with protease type I, whereas trypsin has littleeffect on the anti-coagulant after an incubation period of 3 hours (FIG.11). The protease type I and the trypsin concentration used in theseexperiments, completely inactivate 2.5 nM thrombin in 15 minutes. Theamounts of protease type I and trypsin, carried over from the reactionmixtures to the MPTT, have no effect on the control clotting time.

The MPTT is prolonged in the presence of the anti-coagulant (FIG. 12)both when initiated with HTP and when started with factor X_(a).Thrombin-induced coagulation, however, is not inhibited.

Because of these findings, we investigated the effect of theanti-coagulant on the conversion of prothrombin to thrombin by factorX_(a), factor V_(a), phospholipid and Ca²⁺. Under the experimentalconditions mentioned, thrombin formation is inhibited by theanti-coagulant in a dose-dependent way (FIG. 13A). The activation ofprothrombin by factor X_(a), phospholipid and Ca²⁺ in the absence offactor V_(a) can be inhibited also by the anti-coagulant (FIG. 13B).However, this inhibition is not observed if the activation takes placein the absence of phospholipid (FIG. 13C).

EXAMPLE 5 Polyclonal Antibodies Against VAC

Polyclonal antibodies against bovine VAC were raised in a rabbit. BovineVAC, purified according to the method as described in Example 1, wasmixed with equal amounts of complete Freund's adjuvant. The mixture wasinjected subcutaneously into a rabbit. After a period of 4 weeks, therabbit was re-injected subcutaneously with purified bovine VAC. Thesubcutaneous injections were repeated twice at two-week intervals. Tendays after the last injection, the rabbit was bled and the collectedblood was allowed to clot in order to obtain serum.

Immunoglobulins (Ig) were isolated from the serum according to thefollowing method:

(a) The serum was heated for 30 minutes at 56° C.

(b) Subsequently, the serum was applied to DEAE-Sephacel, which wasequilibrated with 50 mM Tris, 100 mM NaCl, pH 8.2.

(c) The non-bound protein was precipiptated with (NH₄)₂ SO₄ at 50%saturation.

(d) The precipitated proteins were pelleted by centrifugation and thepellet resuspended in 50 mM Tris, 100 mM NaCl, pH 7.9 and dialyzedextensively against the same buffer.

(e) The resulting protein mixture contained anti-VAC Ig.

Following the procedure as described, protein fractions which expressVAC-activity were isolated from bovine aorta, bovine lung, rat and horseaorta, and human umbilical cord arteries.

The proteins were separated by electrophoresis on a polyacrylamide gelin the presence of dodecyl sulfate and under non-reduced conditions.After completion of the electrophoresis, the proteins were transferredfrom the gel to nitrocellulose sheets as described by Towbin et al.,Proc. Natl. Acad. Sci., USA, 76: 4350-4354 (1979). The sheets wereincubated with the anti-VAC Ig and after thorough washing the sheetswere incubated with goat anti-rabbit Ig coupled to horseradishperoxidase. The latter was visualized with the peroxidase substratediamine bezidine tetrahydrochloride.

A brown band on the nitrocellulose sheet, after completion of thedescribed procedure, indicated the presence of goat anti-rabbit Ig.Furthermore, on this spot were present anti-VAC Ig and proteins to whichthe anti-VAC Ig was bound.

Immunoblots of proteins with VAC activity, isolated from bovine aorta,bovine lung, rat and horse aorta, and human umbilical cord arteries arepresented in FIG. 14.

These results show that by essentially using the isolation procedure asdescribed, a protein fraction with VAC activity can be obtained frombovine aorta, bovine lung, rat and horse aorta, and human umbilical cordarteries. Moreover, the isolated protein fractions with VAC activitycontain proteins, with MW of approximately 32,000, 34,000, and 70,000,that react with anti-VAC Ig raised against purified bovine VAC inrabbits.

EXAMPLE 6 Purification of VAC, Using Large Volume Phospholipid Vesicles

Large volume phospholipid vesicles (LVV), composed of1,2-dioleoyl-sn-glycero-3-phosphoserine (PS) and1,2-dioleoyl-sn-glycero-3-phosphocholine (PC), were prepared by themethod of P. van de Waart et al., Biochemistry, 22: 2427-2432 (1983).

For the purification step, LVV containing PS/PC (molar ratio 20:80) wasused. Other molar ratios can be used as long as negatively chargedphospholipids are present. The chain length of the fatty acids in thephospholipids can also be varied.

LVV, ±1 mM phospholipids in 50 mM Tris/HCl, 100 mM NaCl, pH 7.9, weremixed with an equal volume of a protein fraction containing VACactivity. The proteins were in 50 mM Tris/NaCl, 10 mM CaCl₂, pH 7.9. Themixture was allowed to stand for 5 minutes at ambient temperature.Subsequently, the mixture was centrifuged for 30 minutes at 20,000×g.The pellet was resuspended in 50 mM Tris/HCl, 100 mM NaCl, 10 mM CaCl₂,pH 7.9, and recentrifuged. The resulting pellet was then resuspended in50 mM Tris/HCl, 10 mM ethylenediamine tetraacetic acid (EDTA), pH 7.9,and recentrifuged. The resulting supernatant contained the VAC activity.

The above described procedure is an efficient purification step in theprocedure to obtain purified VAC.

                  TABLE A                                                         ______________________________________                                        Summary of the Purification                                                   of VAC from Inner Coat of Bovine Aorta                                                                  Specific                                            Purification                                                                           Protein.sup.a                                                                          VAC.sup.b                                                                             Activities                                                                           Yield Degree of                              Step     mg       Units   units/mg                                                                             %     Purification                           ______________________________________                                        Supernatant                                                                            630      19.000  31.0   100   1.0                                    liquid                                                                        with 35%                                                                      (NH.sub.4).sub.2 SO.sub.4                                                     Precipitate                                                                            470      19.000  40.4   97    1.3                                    with 90%                                                                      (NH.sub.4).sub.2 SO.sub.4                                                     Hydroxy- 206      17.300  84.0   89    2.7                                    apatite                                                                       fraction                                                                      DEAE     35.8     13.900  388    71    12.5                                   fraction                                                                      Sephadex 0.45     0.666   1480   3.4   47.7                                   G-100                                                                         fraction 139                                                                  ______________________________________                                         .sup.a Protein was determined using the method of Lowry et al. (J. Biol.      Chem., 193: 265 (1951).                                                       .sup.b The VAC units were determined using the onestage coagulation test      described in Example 1 by a series of test dilutions. The coagulation tim     of the control samples was 65 seconds. One unit of VAC activity was           defined as the quantity of VAC wh ich prolongs the coagulation time to 10     seconds.                                                                 

                  TABLE B                                                         ______________________________________                                        Cation-Dependent Binding of VAC                                               to Negatively Charged Phospholipid Liposomes                                                 t.sub.c, seconds.sup.a                                                        Supernatant                                                    Cation (10 mm) Liquid.sup.b                                                                             EDTA.sup.c                                          ______________________________________                                        Control (no    180        .sup. N.D..sup.d                                    liposomes)                                                                    Control (no    174        64.8                                                cation)                                                                       CaCl.sub.2     64.2       134                                                 MgCl.sub.2     165        N.D.                                                MnCl.sub.2     65.1       N.D.                                                ______________________________________                                         .sup.a The coagulation time (t.sub.c) was determined using the onestage       coagulation test described in Example 1.                                      .sup.b 50 ul phospholipid liposomes (PS/PC; 50/50 mol/mp: 1 mm), 50 ul VA     (250 ug/ml, specific activity = 700 units per mg), and 100 ul of TBS          containing 5% glycerol and cation, pH 7.5, were mixed at ambient              temperature and centrifuged for 15 m inutes at 15,000 xg. Supernatant         liquid (25 ul) was diluted with TBS to a final volume of 175 ul and teste     using the onestage coagulation test. The remainder of the supernatant         liquid was analyzed with SDSPAGE (FIG. 4).                                    .sup.c The liposome precipitate was suspended in 150 ul of TBS containing     5% glycerol and 10 mm EDTA, pH 7.5. The suspension was centrifuged for 15     minutes at 15,000 xg. The VAC activity of the supernatant was analyzed as     described above.                                                              .sup.d N.D. = not determined.                                            

                  TABLE C                                                         ______________________________________                                        Effect of VAC on the                                                          Amidolytic Activity of Factor X.sub.a and Factor II.sub.a                                   VAC (A.sub.405 /min × 10.sup.3).sup.a                                   -       +                                                       ______________________________________                                        X.sub.a         110.5     110.5                                               X.sub.a, AT-III 80.0      81.5                                                X.sub.a, Heparin                                                                              110.5     109.0                                               X.sub.a, Heparin, AT-III                                                                      47.5      .sup. N.D..sup.b                                    II.sub.a        7.5       7.5                                                 II.sub.a, AT-III                                                                              5.4       5.6                                                 II.sub.a, Heparin                                                                             7.5       7.1                                                 II.sub.a, Heparin, AT-III                                                                     0.56      N.D.                                                ______________________________________                                         .sup.a The amidolytic activity was measured as follows: Factor X.sub.a or     Factor II.sub.a was diluted with the abovementioned agents in TBSA. The       reaction mixture was stirred with a Tefloncoated stirrer in a plastic dis     (37° C.). After 10  minutes, a sample of 100 ul (X.sub.a) or 50 ul     (II.sub.a) was placed in another plastic dish (37° C.) which           contained 800 ul of TBSE, 100 ul of TBSA, and 100 ul of S 2337 (2 mM) or      900 ul S 2238 (5 mM). The change in absorption at 405 nM was mea sured        using a Kontron Spectrophotometer Uvikon 810 (37° C.). The final       concentrations of the various agents in the reaction mixtures were as         follows: Factor X.sub.a (18.7 nM); Factor II.sub.a (1.5 nM); human ATIII      (18.7 nM); heparin (1 unit per ml); and VAC (10.7 ug/ml, specific             activity: 1300 units/mg).                                                     .sup.b N.D. = not determined                                             

What is claimed as new and intended to be secured by Letters Patent is:
 1. A process for preparing an anti-coagulant protein from tissue which comprises:(a) homogenizing said tissue, differentially centrifuging said homogenized tissue, and subjecting the supernatant liquid to one or more of the following purification treatments in any desired sequence: (b) precipitation with salt, (c) affinity chromatography, (d) ion exchange chromatography, (e) chromatography using a molecular sieve;wherein said tissue is selected from the group consisting of blood vessel walls, highly vascularized tissue, or endothelial cell cultures.
 2. The process of claim 1 wherein said anti-coagulant protein is further purified using immunoabsorption chromatography.
 3. The process of claim 1 wherein said anti-coagulant protein is purified using phospholipid vesicles.
 4. The process of claim 1 wherein ammonium sulfate is used for the precipitation in step (b), hydroxyapatite is used for chromatography in step (c), DEAE-Sephacel is used for chromatography in step (d), and Sephadex G-100 or G-75 is used for chromatography in step (e). 